The interaction between human endothelial (EC) and adipose mesenchymal stem/stromal cells (ASC) leads to EC reorganization into stable vascular structures in vitro and functional vessels in vivo and ASC maturation from progenitor to smooth muscle/pericyte phenotype. We have previously shown that juxtacrine EC-ASC interactions induce in ASC to express activin A, which, by affecting both EC and ASC bioactivities, plays a central role in vasculogenesis. Here, a co-culture model of human cord blood-derived EC and human ASC was used to study the role of Notch2 and Notch3 in juxtacrine mechanism-mediated regulation of activin A expression in ASC. Blockade of Notch signaling by the γ-secretase inhibitor DAPT, between days 1-3 following heterocellular contact, prevented induction of activin A secretion, and DAPT added to co-cultures after establishment of activin A secretion almost completely abrogated its production, suggesting the need for uninterrupted Notch signaling to enforce continued activin A production. In parallel, DAPT reduced ASC differentiation towards the mural phenotype, and increased EC proliferation, causing inefficient vasculogenesis. Silencing Notch2 expression in ASC prevented activin A production by ASC when in co-culture with EC, whereas silencing Notch3 expression amplified its secretion. Dual silencing experiments revealed that silencing Notch2 was dominant, eliminating the effect of silencing Notch3. In parallel, Notch2 was not involved in mediating differentiation of ASC towards a smooth muscle/pericyte fate, whereas silencing of Notch3 led to an increase in baseline expression of markers of smooth muscle phenotype, which was further enhanced on contact with EC. Overall, Notch3 inhibits both expression of activin A and smooth muscle/pericyte differentiation program. Furthermore, these data suggest that activin A is induced exclusively through trans signaling mechanisms, whereas mural differentiation program is also regulated through cis pathways.